The application proposes to continue examining the signal transduction processes transmitting death signal to the cardiomyocytes during myocardial ischemia and reperfusion. During the current years' funding, our laboratory has documented the importance of MAP kinase pathway and its regulation by redox signaling. A recent study from our laboratory has demonstrated that sphingomyelin, which comprises 10-15% of the total phospholipids content, is an important signaling molecule that plays a crucial role in myocardial ischemic reperfusion injury. Ceramide, generated from the breakdown of sphingomyelin, can however be involved in ischemic preconditioning by triggering a survival signal through the generation of sphingosine-1-phosphate. The proposed research will attempt to resolve the paradoxical role of ceramide signaling, which can generate both death and survival signals by addressing four Specific Aims: i) by determining the mechanism of the formation of ceramide from Sphingomyelin breakdown leading to the development of cardiac dysfunction during ischemia/reperfusion; ii) by examining if preconditioning-mediated cardioprotection is due to reduction of ceramide formation causing upregulation of sphingosine-1-phosphate leading to the upregulation of nitric oxide production; iii) by studying whether ceramide generated in the ischemic reperfused heart becomes associated with the "lipid rafts" causing their stabilization, leading to the formation of large raft domains ("platforms"), which then transmits death signal in the ischemic heart and survival signal in the preconditioned heart; and iv) by determining if the "lipid rafts" control the nitric oxide signaling thereby modulating ischemia/ reperfusion-induced "death signal" into preconditioning-induced "survival signal". The overall objective is to delineate the dichotomy in the behavior of ceramide signaling in generating both death and survival signal during ischemia/reperfusion and preconditioning, respectively, and to develop therapeutic modalities to reduce the ischemia/reperfusion-mediated death signal by modulating lipid raft-associated nitric oxide signaling. [unreadable] [unreadable]